The bacterial siderophore enterobactin confers survival advantage to Salmonella in macrophages

Abstract

Enterobactin (Ent), a prototypical bacterial siderophore known for its unparalleled affinity for iron, is widely conserved among members of the Enterobacteriaceae family of Gram-negative bacteria. In this study, we demonstrated that, aside from mediating iron acquisition, Ent also dampened the macrophages (MΦs) antimicrobial responses against intracellular infection by Salmonella enterica serovar Typhimurium. Accordingly, the loss of Ent expression (ΔentB) in Salmonella demoted their survivability against MΦs. Addition of exogenous Ent not only rescued the survival of ΔentB Salmonella, but also augmented WT Salmonella to better withstand the microbicidal activity of MΦs. The protection conferred to WT Salmonella was observed only when Ent was administered as iron-free, thus indicating the requirement of iron chelation in this context. In contrast, the exogenous iron-bound Ent retained its ability to promote the survival of ΔentB Salmonella, albeit modestly. Assessment on MΦs labile iron pool (LIP) revealed that iron-free Ent is able to permeate into MΦs, chelate the intracellular LIP, and regulate the expression of several key iron-regulatory proteins, i.e., divalent metal transporter 1, ferroportin, and hepcidin. Chelation of iron by Ent was also observed to promote the MΦs towards M2 polarization. Collectively, our findings demonstrated that Ent not only facilitates bacterial iron uptake but also disrupts MΦs iron homeostasis and M1/M2 polarization to safeguard intracellular bacteria against the anti-bacterial effects of their host.

Keywords: Enterochelin; iron; iron chelation.

Figure 1.

Ent provides an intracellular survival advantage to Salmonella. Production of Ent by Salmonella and its isogenic mutants (ΔentB) were assessed using CAS assay. (a) The formation of orange halo indicates siderophore production by WT Salmonella and ΔentB mutant on CAS agar plate. (b) Bar graphs indicate the relative siderophore activity in the bacterial culture supernatant detected via CAS liquid assay. (c) BMDMs were pre-incubated with Ent, then infected with WT Salmonella or ΔentB mutant at MOI of 100, and treated with Ent for 24h in presence of gentamicin (20 µg/ml). Intracellular bacterial survival was determined by serial dilution and plating on LB plate. The impact of Ent on the intracellular survival of WT Salmonella activated with IFN-γ (20 ng/ml) or L-arginine (500 µM). (d) BMDMs were infected with WT Salmonella or ΔentB mutant for 24h in presence of gentamicin. (e) MΦs were pre-incubated with Ent, FeCl_3, or Ent+ FeCl₃ (1:1 ratio, 1h) then infected with WT Salmonella or ΔentB mutant and treated with Ent, FeCl₃ or Ent+ FeCl₃ for 24h in presence of gentamicin. In vitro assays were performed in triplicate and data represented as mean ± SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 2.

Exogenous ent upregulates iron uptake regulatory genes in ent-deficient Salmonella. The quantitative RT-PCR analysis was used to quantify bacterial (WT Salmonella and mutant ΔentB) mRNA expression of (a) TonB, (b) FepA and (c) FeS upon treatment with Ent or Ent+ FeCl₃ (1h). mRNA values are represented as fold-change normalized to the Gyrase B housekeeping gene. In vitro assays were performed in triplicate and data represented as mean ± SEM. * p < 0.05, and *** p < 0.001.

Figure 3.

Ent polarizes the Salmonella-infected M1-MΦs towards the M2 phenotype. Mice BMDMs were pre-treated with or without Ent, and then infected with Salmonella (MOI of 100). After washing, cells were incubated for 18h in presence of Ent. Cells were stained with anti-mouse F4/80, CD38, and CD206 antibodies. (a) F4/80⁺ MΦs were gated and analyzed for % positivity of F4/80⁺CD38⁺ CD206⁻ (M1-MΦs) and F4/80⁺ CD38⁻CD206⁺ (M2-MΦs). (b and c) Bar graphs represented the % positivity of F4/80⁺CD38⁺CD206⁻ and F4/80⁺ CD38⁻CD206⁺ cells in basal and Salmonella infected MΦs treated with Ent. In vitro assays were performed in triplicate and data represented as mean ± SEM. ** p < 0.01, and *** p < 0.001.

Figure 4.

Ent disrupts the intracellular iron pool (LIP) in MΦs. (a) Control and (b) Salmonella-infected BMDMs were incubated with Ent (25 µM) and cytosolic LIP was quantitated using the Calcein-AM method. Histograms represent the flow cytometric measurement of LIP in control and Salmonella-infected BMDMs, quantified as mean fluorescence intensity (MFI). (c) Bar graphs represent the LIP chelated by Ent in control and Salmonella-infected MΦs. In infection assay, BMDMs were pre-incubated with Ent then infected with Salmonella (WT strain or its isogenic Ent mutant ΔentB) at MOI of 100 and treated with Ent for 8h in presence of gentamicin (20 µg/ml). Cells were harvested and quantitative RT-PCR analysis was used to quantify mRNA expression of (d) DMT1 (e) Fpn1, and (f) Hamp. Values are represented as fold-change normalized to 36B4 housekeeping gene. In vitro assays were performed in triplicate and data represented as mean ± SEM. * p < 0.05 and ** p < 0.01.